We have previously found that TJ-48 has the capacity to accelerate recovery from hematopoietic injury induced by radiation and the anti-cancer drug mitomycin C (MMC). The effects are found to be due to its stimulation of spleen colony-forming unit (CFU-S) counts on day 14. In the present study, we attempt to isolate and purify the active components in TJ-48 extracts using a new in vitro hematopoietic stem cell (HSC) assay method. n-Hexane extract from TJ-48 shows a significant stimulatory activity. The extract is further fractionated by silica gel chromatography and HPLC in order to identify its active components. 1H-NMR and GC-EIMS indicate that the active fraction is composed of free fatty acids (oleic acid and linolenic acid). When 27 kinds of free fatty acids (commercially available) are tested using the HSC proliferating assay, oleic acid, elaidic acid, and linolenic acid are found to have potent activity. The administration of oleic acid to MMC-treated mice enhances CFU-S counts on days 8 and 14 to twice the control group. These findings strongly suggest that fatty acids contained in TJ-48 actively promote the proliferation of HSCs. Although many mechanisms seem to be involved in the stimulation of HSC proliferation, we speculate that at least one of the signals is mediated by stromal cells, rather than any direct interaction with the HSCs.
"Juzen-Taiho-To" (TJ-48), which is a kampo (Japanese herbal) medicine prepared by decocting a prescription of ten kinds of herbs, has several immunostimulating activities. In order to characterize the active substances for anti-complementary and mitogenic activities, TJ-48 was fractionated. Anti-complementary activity was observed in the water- and methanol-insoluble fraction (F-2) and the crude polysaccharide fraction (F-5), whereas mitogenic activity was only found in F-5. However, other low molecular mass fractions did not show both activities. Methylation analysis indicated that F-2 mainly contained amylopectin-like polysaccharides. Both Pronase digestion and periodate oxidation decreased the anti-complementary activity of F-2, and the beta-amylase-resistant fraction of F-2 still retained the potent anti-complementary activity. When F-5, which has the most potent of both activities, was further fractionated, only the major acidic polysaccharide fraction, F-5-2, showed potent mitogenic activity. Endo-alpha-(1----4)-polygalacturonase digestion showed that F-5-2 mainly contained pectic polysaccharides, and the endo-polygalacturonase treatment of F-5-2 reduced the mitogenic activity but not the anti-complementary activity. F-2 and F-5 each activated the complement system by a different mode of action.
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